The importance of new anti-infective strategies cannot be overstated. Emergence of new conditions caused by previously unknown infectious agents, in combination with the rapid increase in known and novel antimicrobial-resistant pathogens, has led to crises in therapeutic options that require new ways of thinking. One approach to this problem is the development of novel anti-infective compounds that can address aspects of the problem. Our laboratories have identified and characterized a novel thiourea-based small molecule, termed ST-669 that has low-micromolar activity against a wide variety of viruses as well as the intracellular bacteria Chlamydia and Coxiella burnetii. Viruses sensitive to the action of ST-669 include vaccinia (EC-50 = 0.25 micromolar), ebola, influenza, HIV-1, dengue, encephalomyocarditis virus, and Lacrosse virus. (EC-50 = 0.27 micromolar). The overall goal of this project is to identify the mechanism of action of ST-669. The compound is active only in cells of primate origin and activity is sensitive to treatment of cells with cycloheximide. The collected data surrounding ST- 669 activity lead us to hypothesize that the compound works by upregulating a host protein or metabolic pathway. Therefore, our two aims use complementary approaches to knock out (Aim 1) or knock down (Aim 2) all possible genes in cells of human origin and looking for individual mutations that abrogate the inhibitory effects of the compound. In Aim 1, a haploid human cell line will be randomly mutated using a retroviral gene trap approach and the collected mutated populations screened for elimination of ST-669 activity. In Aim 2, we will use a global siRNA library in a high throughput screening strategy to knock down individual protein abundance, and examine treated cells for lack of ST-669 activity. Growth of Chlamydia caviae will be used in this screening approach, in a high throughput assay routinely used in the laboratory. Secondary screens for each assay will be conducted and the resulting data examined for consistencies and inconsistencies. These results will position our laboratories to develop hypothesis-driven experiments to elucidate the specific mechanism of action of ST-669, and to design approaches to target this pathway in future small molecule screens. These experiments may also identify novel tools used by cells to control infections by very different groups of intracellular pathogens.